The formation of slow and fast twitch fibers in skeletal muscle is a hallmark of functional maturity, enabling muscles to respond to complex physiological demands. Although striated muscles are exquisitely designed to execute diverse physical and metabolic tasks beginning immediately after birth, an understanding of how slow and fast skeletal muscle fibers initially form during fetal skeletal muscle development is still limited. It is our long-term goal to uncover the mechanisms of fiber type differentiation in the fetal skeletal muscle. Our recent data suggest that during fetal skeletal muscle development, Sox6 is required for normal fiber type-specific gene expression. In Sox6-null skeletal muscle, suppression of slow fiber-specific gene expression fails to occur in late fetal stages and slow fiber-specific genes maintain high levels of expression in the future fast fiber muscles, even in the postnatal stages. Sox6 is a member of the Sox transcription factor family. Although it is highly expressed in skeletal muscle, Sox6's role in muscle development is unknown. In this proposal, we will investigate the role of Sox6 in fiber type-specific gene expression during the fetal skeletal muscle development. Our central hypothesis is that Sox6 functions as a transcriptional repressor of slow fiber type-specific genes in the future fast fibers of the fetal skeletal muscle. The three aims proposed to test our hypothesis are: Aim1. Test the hypothesis that Sox6 expression in muscle is required for the initial fiber type differentiation in fetal skeletal muscle. Aim 2. Determine the mechanisms that regulate fiber type-specific Sox6 activity by testing two hypotheses: modulations by protein-protein interactions and post-translational modifications. Aim 3. Test the hypothesis that Sox6 coordinately regulates transcription of multiple genes which specify fiber types by: (1) identifying primary Sox6 targets using ChIP on chip assays and (2) determining how Sox6 represses the MyHC-2 gene These aims will be achieved by determining the phenotype of skeletal muscle specific Sox6 conditional knockout mice (Aim 1), by identifying Sox6 co-factors using yeast two-hybrid assays and determining post-translational modification of the Sox6 protein in skeletal muscle (Aim 2), and by identifying primary targets of the Sox6 protein using ChIP on chip assays and determining the molecular mechanism of transcriptional suppression of the MyHC-2 gene by Sox6. Completion of the proposed aims will advance our knowledge in the terminal differentiation of fetal skeletal muscle and aid us in gaining important information for treating muscle degenerative diseases. Project Narrative In this A1 resubmission, we investigate the role of the Sox6 transcription factor in the fetal muscle fiber type specification. Our central hypothesis is that Sox6 functions as a transcriptional repressor of slow fiber type-specific genes in the future fast fibers of the fetal skeletal muscle. To test this, we will ask following questions in three specific aims: when and where, during myogenesis, does Sox6 function? (Aim 1);what are the cellular mechanisms to limit the repression of slow fiber type-specific genes by Sox6 to fast fibers? (Aim 2);and finally, how does Sox6 achieve transcriptional suppression of slow fiber type-specific genes at the molecular level? (Aim 3).